In general, a protein production system utilizes microorganisms such as Ecolab to which a gene encoding desired protein is transfected, expressing said protein. However, the production of conjugated proteins (protein with sugar chain) is difficult with a microorganism due to its cellular structure. There are also undertakings for producing conjugated proteins that employ yeast and some animal cells (hamster cells). However, their problem is that the protein that is obtained has a glycosylation that is specific to the cells involved in the production, thus differing from the conjugated proteins originated from humans, which are desired for medical treatment.
At research levels, there are reports of producing protein by transfecting genes to human cells; however, the production period is at the level of approximately one month, thus being a transient result. So far, there is no example of success with a long and stable production period for industrial use.
In the other words, the following problems exist in the above-mentioned conventional methods:
First, a protein producing system utilizing prokaryotic cells such as a microorganism can produce protein with simple structure only, such as a part of the site of action (including the active center) in an enzyme. It is considered that since the intracellular protein synthetic pathways of microorganisms are different from that of higher animal cells, it is difficult to form tertiary structures of large molecular weight proteins.
In addition, in the case of conjugated proteins in which proteins are coupled with a substance other than protein such as sugar chain, since a microorganism itself does not have organs for synthesizing conjugated proteins, it is impossible to synthesize conjugated proteins in principle.
Also, unlike the case of the above-mentioned microorganism, protein production which utilizes yeast, animal cells other than that of humans, or insect cells, possesses organs to synthesize conjugated proteins within cells. Therefore, they can synthesize high levels of proteins. However, said proteins are modified with unique sugar chains derived-yeast or -heterologous cells other than that of humans. Thus it is difficult to obtain human-derived gene products.
Such problems can be solved by employing human cells (a higher animal's cells), to which a gene encoding a desired protein is transfected, and expressed. However, besides some examples of transient expression, problems remain. Such problems include the difficulty of maintaining a long-term protein production over one year, which would allow for industrial production; the loss of protein productivity within 2 months post transfection; and the smallness of expressed protein quantities.
For these problems, a method by the activation of endogenous genes in human cell strains is proposed as a way to produce conjugated protein of human origin (JP-T 2001-511342 specification (patent document 1)). However, because this technique is a production of human protein in a human cell strain by means of activating an endogenous gene, a human cell strain has to be selected according to a target protein. Thus such selection becomes an issue. Moreover, the production efficiency is unstable because it varies according to the protein to be produced. Consequently its efficiency ends up being low, the majority of the time. Furthermore, its applicability is low, because it cannot produce conjugated protein other than that of human origin utilizing the aforementioned human cell strain and due to other reasons.
A conjugated protein production method that addresses the above problems and can realize a long term and stable production of such proteins is therefore highly desired.
Accordingly, this invention aims to offer a novel human cell strain, wherein a transformed human cell strain is used to realize a long term and stable production of protein originated from a gene by means of gene transfection; a method of selecting the novel human cell strain; use of the novel human cell strain; a protein producing method utilizing the novel human cell strain; and a pharmaceutical composition utilizing the novel human cell strain.
In addition, there exist the following prior art literature related to the present invention in addition to the patent document 1 described above: Japanese Patent Laid-Open No. 2002-51780 (Patent document 1); JP-T 2001-500381 (Patent document 2); Japanese Patent laid-Open No. 8-163982 (Patent document 3); JP-T 2003-509025 (Patent document 4); Japanese Patent Laid-Open No. 5-310795; (Patent document 5); Japanese Patent Laid-Open No. 6-141882 (Patent document 6); JP-T 8-501695 (Patent document 7); Japanese Patent Laid-Open No. 6-78759 (Patent document 8); Japanese Patent Laid-Open No. 2003-274963 (Patent document 9); Japanese Patent Laid-Open No. 2002-58476 (Patent document 10); JP-T 2000-506379 (Patent document 11). However, none of these prior art disclosures achieve the abovementioned objective of this invention.